The present invention relates to a new hyperosmotic intracellular flush and storage solution for preserving an organ for transplantation. In addition, the present invention relates to a method for preserving an organ to be transplanted by using an adenosine-MgSO.sub.4, mannitol intracellular flush solution.
While the invention shall be described in connection with the preservation of kidneys, it is understood by those skilled in the art that the intracellular flush and storage solution disclosed herein and the preservation method utilizing the same are applicable to other organs such as the pancreas, the liver, and the heart.
Recently, a great deal of progress has been achieved in the field of organ transplantation through the use of cyclosporine A. Cyclosporine A is a powerful immunosuppressive drug which appears to act mainly on T cells. Through the use of cyclosporine A, a 20% increase in one year allograft survival of kidneys has been noted over that of conventional therapy. However, this advantage appears to be lost with increasing preservation times of the organs to be transplanted (Opelz, G.: Multicenter Impact of Cyclosporin on Cadaver Kidney Graft Survival, Prog. Allergy 38: 329-345, 1986). In addition, there is increasing evidence that moderate ischemic injury based upon unsatisfied metabolic oxygen demand may predispose renal allografts to severe rejection and diminished survival (Keller, H., Fischer, G., Kirste, G., Wilms, H.: ATN Influence on Renal Transplant Function, Transpl. Proc. (in press) 1989). Thus, in today's cyclosporine era, better preservation techniques are essential for optimal allograft survival, not just to diminish the detrimental effects of prolonged preservation times, but also to prevent the occurrence of delayed graft function which is associated with further graft loss using cyclosporine prior to the complete resolution of post renal transplant acute tubular necrosis (Bia, M. J., Tyler, K. A.: Effect of Cyclosporine on Renalischemic Injury, Transplantation, 43:800-804, 1987).
In this regard, a great deal of research progress has been made over the years in understanding cellular mechanisms, as well as developing new preservation techniques for keeping kidneys and other organs viable not only during cold storage, but also after revascularization and reperfusion of these organs. Early investigators were mainly interested in studying physiologic characteristics of isolated organs. Their methods allowed only very short observation periods before the organs suffered irreversible ischemic damage due to the lack of blood supply. However, in 1938, Carrel and Lindbergh contributed substantially to the knowledge in organ preservation by showing that kidneys could be kept viable extracorporeally for a limited time by using a special blood perfusion apparatus (Carrel, A. and Lindbergh, C. A.: The Culture of Organs, New York, Paul B. Hoeber, Inc., 1938). Later, profound hypothermia (i.e. a much lower than normal temperature) was found to prolong the period in which tissue could tolerate ischemia.
Moreover, successful cadaver kidney transplantation in the early 1960's greatly stimulated further work in the field of renal preservation. In 1963, Humphries and co-workers reimplanted dog kidneys after 24 hours of extracorporeal hypothermic perfusion with diluted serum or plasma (Humphries A. L., et al.: Successful Reimplantation of Canine Kidney After Twenty Four Hour Storage, Surgery, 54:136, 1963). Belzer and colleagues, in 1967, achieved a very significant breakthrough by preserving dog kidneys for as long as 72 hours using hypothermic pulsatile perfusion with cryoprecipitated plasma (Belzer, F. O., Ashby, B. S., Dunphz, J. E.: Twenty Four Hour and Seventy Two Hour Preservation of Canine Kidneys, Lancet 2: 536, 1967). This was very quickly followed by consistently successful human cadaver kidney preservation using Belzer's method. Subsequently, Johnson, et al. transplanted canine kidneys preserved with pulsatile perfusion but employing plasma protein fraction (PTF) as the perfusate (Johnson, R. W. G., et al.: Evaluation of a New Perfusion Solution for Kidney Preservation, Transplantation, 13:270, 1972), while Claes and associates introduced 4.5% human albumin solution as a kidney preservation perfusate (Claes, G., et al.: Albumin as Perfusate in Continuous Perfusion for Renal Preservation, Fourth International Transplant Conference, New York, Grune and Stratton, p. 46, 1972). In 1969, Collins and colleagues had further simplified the preservation technique by showing that ample storage of dog kidneys in ice slush following immediate initial flushing with an intracellular electrolyte solution was successful in preserving the kidney for as long as 30 hours (Collins, G. M., Bravo-Sugarma, M., and Terasaki, P.: Kidney Preservation for Transplantation, Lancet, 2:1219, 1969).
Today, both simple cold storage and continuous pulsatile perfusion are used in clinical renal transplantation, either separately or in combination. Simple cold storage is used more extensively because it is generally accepted that a human cadaver kidney can be safely preserved by the simple cold storage method when the kidney has sustained only minimal ischemia and can be implanted within 40 hours. Otherwise, hypothermic pulsatile perfusion has been recommended.
Simple cold storage combines the hypothermic and flush solution effects to decrease metabolic activity and prevent subsequent cell swelling and acidosis. Simple cold storage is accomplished by rapidly cooling the kidney immediately after harvesting by flushing the renal vasculature with chilled electrolyte solution and then placing the kidney in ice slush. The core temperature is kept between 0.degree. and 4.degree. C. by placing the sterile sealed kidney container in ice, where it is kept until the cadaver transplant procedure can be performed. This method of preservation is also employed for most extracorporeal renal operations.
Although simple cold storage methods have offered consistently successful preservation of kidney viability up to 48 hours (Halasz, N. A., Collins, G. M.: Forty-Eight Hour Kidney Preservation: A Comparison of Flushing and Ice Storage with Perfusion, Arch. Surg. 111:175-177, 1976), this time period becomes more critical if the cadaver donor was poorly prepared prior to donor nephrectomy or if the kidney has sustained a period of warm ischemia. In these situations, hypothermic pulsatile perfusion after renal flushing with a chilled electrolyte solution is the preferred method of renal preservation. An advantage of this method is a longer safe preservation period, in some cases as long as 72 hours. Most transplantation centers are employing human cadaver kidneys that have been preserved for longer periods than in the past, due to an increase in regional and national organ sharing. These kidneys can be readily transported in a portable pulsatile renal preservation unit. Compared to simple hypothermia, pulsatile perfusion is technically more complex and more costly (Alijani, M. R., et al.: Single-donor Cold Storage vs. Machine Perfusion in Cadaver Kidney Preservation, Transplant 40:659-661 1985), however, it does seem to offer more reliable preservation in the 48 to 72 hour range, thus this type of preservation is often used for questionably viable kidneys obtained from inadequately prepared cadaveric donors or rare AB kidneys, which may travel through several centers prior to matching with an appropriate recipient.
The ability to successfully preserve the kidney for as long as 72 hours has been of immense benefit to cadaver renal transplantation. This has provided sufficient time for both histocompatibility testing of the donor and sensitive cross match testing for preformed cytotoxic antibodies in the recipient or for organ sharing between transplant centers. However, notwithstanding the above, improved preservation techniques, including enhanced intercellular flush solutions, are needed not only to extend the preservation period, but also to improve the quality of the organs transplanted.
To understand existing renal preservation techniques and the rationale for potentially new developments, one must first understand what occurs at the cell level during periharvest warm ischemia, subsequent hypothermic storage (cold ischemia) and reperfusion, such as diminished metabolic activity, as well as cessation of cell membrane function which leads to cell swelling and acidosis. Concurrent with this there is a continued loss of intracellular energy stores which subsequently generates toxic free radicals, contributing to further endothelial damage or "reperfusion" injury after revascularization upon transplantation.
More particularly, as a result of the deprivation of circulation, and thus oxygen (i.e. ischemia), during transplantation, the sodium pump of the renal cells, which normally maintains the intracellular composition of the renal cells high in potassium, magnesium, and phosphate and low in sodium and chloride, ceases to function due to the lack of energy, resulting in an inflow of sodium and chloride into the cells, and an outflow of potassium and to a lesser extent magnesium from the cells (Sacks, S. A., Petritsch, P. H., Kaufman, J. J.: Canine Kidney Preservation Using a New Perfusate, The Lancet, May 12, 1973; 1024-1028). The result of these rapid changes in ion distribution in the ischemic cell is a net gain, not merely an exchange, of intracellular ions (sodium and chloride) followed passively by water (Leaf, A. Ann. N.Y. Acad. Sci., 72, 396, 1959) and a profound loss of potassium and to a lesser extent magnesium (Keeler, R., Swinney, J., Taylor, R. M. R., Uldall, P. R., Br.J. Urol., 38, 653, 1966). Moreover, the remaining non-diffusible intracellular molecules (with electronegative charge) exert Donnan and osmotic forces, resulting in the further movement of water into the cell (Leaf, A., Am. J. Med., 40, 291, 1970). The morphological consequence of these changes in ion and water distribution of the renal cells is characterized by "cellular swelling" which impedes the flow of blood through the kidneys. Red blood cells have been shown to impact in the swollen glomerular capillary bed after macrocirculation has been reestablished to kidneys subjected to ischaemia (Summers, W. K., Jamison R. L., Lab. Invest. 25, 635, 1971) thereby markedly reducing the efferent flow to the peritubular capillaries. The ischemic insult to the renal tubular cell may thus be perpetuated even after the macrocirculation has been reestablished (Sacks, supra.).
Moreover, there is increasing evidence that much of the damage resulting from a period of warm or cold ischemia in many organs, including the kidneys, is produced by the continued loss of intracellular energy stores which subsequently generates toxic free radicals which damage cell membranes of the endothelial cells which line the fine microcirculation of the kidney upon transplantation (i.e. reperfusion injury). The mechanism involved in the free radical generation in the ischemic tissues at reperfusion is now thought to be known. Briefly, the high energy phosphate compounds are broken down stepwise (ATP - ADP - AMP) with the onset of ischemia, resulting in the accumulation of hypoxanthine, the concentration of which increases progressively during the period of ischemia. There is also rapid proteolytic conversion of xanthine dehydrogenase to xanthine oxidase during the period of ischemia. Consequently, both activated enzyme (xanthine oxidase) and its oxidizable substrate (hypoxanthine) are present in excess. At the moment of reperfusion, the reducible substrate, molecular oxygen, is supplied suddenly and in excess, leading to a burst of superoxide radical production. Both superoxide anions and their toxic descendants, including hydroxyl radical, can cause tissue injury (Hoshino, T.; Maley, W.; Bulkey, G.; and Williams, G.: Ablation of Free Radical-Mediated Reperfusion Injury for the Salvage of Kidneys taken from Non-heartbeating Donors, Transplantation, Vol. 45, 284-289). Normally, the cytochrome oxidase complex present in the kidneys and other organs can supply enzymes such as supraoxide dismutase and catalase (SOD+CAT) to scavenge these free radicals and rid the cell of these toxins by further degradation. However, with ischemia these are not sufficient to prevent further injury after reperfusion.
In an attempt to deactivate the harmful O.sub.2.sup.- and OH.sup.- free radicals, mannitol (Pavlock, G. S., et al.: Effects of Mannitol and Chlorpromazine on Pretreatment of Rabbits on Kidney Mitochondria Following In Vivo Ischemia and Reflow, Life Sci. 29: 2667-72, 1981), DMSO (Hansson, R., et al.: Effect of Xanthine Oxidase Inhibition of Renal Circulation After Ischemia, Transplant. Proc. 14:51-8, 1982), and L-methionine (Hansson, supra) have been added with only a limited degree of success. Moreover, there has also been much controversy about the role of calcium in ischemic nephrotoxicity. It is postulated that calcium activates various calcium dependent phospholipases which start a cascade of reactions which amplify the destructive effects of ischemia. The cascade responsible for the cell membrane degradation, may be inhibited somewhat by calmodulin inhibitors, calcium entry blockers (CEBs), or Chlorpromazine (Frega, W. S., et al.: Ischemic Renal Injury, Kidney Int. 10:517-25, 1976) (Pavlock, G. S., et al.: Effects of Mannitol and Chlorpromazine Pretreatment of Rabbits on Kidney Mitochondria Following In Vivo Ischemia and Reflow, Life Sci. 29:1667-72, 1981). Thus, it is not only important to understand what is going on in the kidney during cold storage, but also to reassess what occurs in the immediate reperfusion period post renal transplant. In order to counteract the above pathologic processes which occur at the cell level during warm ischemia, subsequent hypothermia storage and reperfusion, cold storage (i.e. hypothermia) and various electrolyte flushing solutions have been used to extend the preservation times of the kidneys to be transplanted. The main function of hypothermia (4.degree.-8.degree. C.) is to slow metabolic activity and intracellular respiration by a factor of 10-20, extending the one hour normothermic (37.degree. C.) limit to 13 hours of cold. By maintaining the kidney in a cold state, the metabolic needs of the kidney are kept to a minimum. Thus, the objective of cold storage is to bring the kidney as rapidly as possible to a temperature close to its freezing point without subjecting it to shock and to maintain the kidney at this low temperature until just prior to transplantation, when its temperature is raised to a level close to normal body temperature.
In order to extend organ preservation by cold storage (hypothermia), various electrolyte flushing solutions have been developed over the years to be utilized in combination with hypothermia. Initially, the principle aims were to achieve rapid cooling and to wash out the blood with its particulate compounds, coagulation factors, and isoaggultinins (Collins, G. M.: Current Status of Renal Preservation by Simple Flushing and Hypothermic Storage, Renal Preservation, Chapter 15, 224-243). Little attention was paid to the precise composition of these solutions which were chosen on the whole from those readily available to clinical use and were thus basically of extracellular composition. (Collins, supra at 226).
In this regard, Ringer's lactate and isotonic saline solutions were determined to be fairly good extracellular flushing solutions that allowed safe renal preservation for short periods of time, i.e. up to four hours. Thus, extracellular solutions may be used in the living renal donor transplant setting for short periods of preservation time, however, longer periods are not advised. For example, the present inventors have observed severe histologic ischemic damage and subsequent nonfunction in dog kidneys preserved for five hours by cold storage after flushing with Ringer's lactate solution.
However, notwithstanding the above, it was discovered that intracellular electrolyte solutions, such as the intracellular electrolyte solution developed by Collins and associates (Collins, G. M., Bravo-Sugerma, M., and Terasaki, P.: Kidney Preservation for transplantation, Lancet 2:1219, 1969) offer several advantages for simple cold storage in comparison with other recently developed intracellular flush solutions. Table 1 below shows the ingredients of the Collins solution (solution C2) most commonly used today in comparison with other recently developed intracellular flush solutions.
TABLE 1 ______________________________________ COMPOSITION OF DIFFERENT INTRACELLULAR RENAL FLUSH SOLUTIONS (g/l) BELZER COLLINS-2 SACKS-2 PERFUS- UW-1 FLUSH FLUSH ATE FLUSH ______________________________________ KH.sub.2 PO.sub.4 2.0 4.16 3.4 3.4 K.sub.2 HPO.sub.4 9.70 9.70 -- -- 3H.sub.2 O KCl 1.12 -- -- -- KHCO.sub.3 -- 2.30 -- -- Mannitol -- 37.5 -- -- Glucose 25 -- 1.5 -- MgSO.sub.4 7H.sub.2 O 7.38 -- 8 1.2 MgCl.sub.2 -- (2 meq/ml) -- -- Adenosine -- -- 1.3 1.34 Sodium -- -- 17.5 0.92 Glutathione Albumin -- -- 5.3 -- NaHCO.sub.3 0.84 1.26 -- -- Allopurinol -- -- 0.113 0.113 Verapamil -- -- -- -- K+-Lacto -- -- -- 39.8 bionate Raffinose -- -- -- 17.8 Hydroxyethyl -- -- -- 50 Starch Osmolality 320 430 300 320- 330 (mOsm/kg) pH 7.00 7.00 7.10 7.40 ______________________________________
In addition, a number of "modified" Collins' solutions have been produced in an attempt to improve the preservation properties (i.e. duration and quality of kidney storage) of the flush solution for simple cold storage. In this regard, Euro-collins solution is similar to Collins' solution except it does not contain magnesium. Despite this, multiple studies have shown equivalent preservation using both types of Collins' flush solutions during simple hypothermic storage. The exact mechanism of action and the significance of the ionic composition of the intracellular flushing solution have been disputed. Belzer and Downes performed interesting laboratory experiments comparing Collins' solution and hyperosmolar Ringer's lactate solution, and concluded that cellular potassium loss during cold storage is not as critical to subsequent renal function as is the prevention of water gain (Belzer, F. O., Downes, G. L.: Organ Preservation for Transplantation, Boston, Little, Brown, 1974). These authors also suggested that the osmotic effect of a high glucose content in Collins' solution was more important than were the high potassium and magnesium concentrations. Nevertheless, it is well accepted that these phosphate buffer solutions are quite effective in preventing cell swelling and acidosis during simple hypothermia storage as previously discussed.
Furthermore, Sacks and his associates developed a number of flush solutions (S.sub.1 and S.sub.2) which reportedly improved transplantation results after storage of kidneys up to 72 hours in a modified perfusion fluid with high intracellular ion concentration and osmotic pressure. (Sacks, S. A., Petritsh, P. H., Kaufman, J. J.: Lancet 1:1024, 1973). Table 1 illustrates the composition of Collins' C-2 solution in comparison with Sacks' S-2. The most significant changes were higher K.sup.+ and lower Na.sup.+ concentrations and the substitution of 37.5 g/liter of mannitol for glucose as t he osmotic active substance thereby increasing the osmolarity from 310 to 430 in Osmol/liter. However, there are many studies that show that the Sacks' solutions exhibit inferior preservation properties when compared to Collins' C-2 solution (Collins, G. M.: Current Status of Renal Preservation by Simple Flushing and Hypothermic Storage, Renal preservation, edited by M. Marberger and K. Dreikorn, Williams & Wilkins, Baltimore, Chapter 15, p. 224-236, 1983) as well as failure to achieve 48 hours of safe preservation adequately (Chatterjee, S. N., Berne, T. V.: Failure of 48 Hours of Cold Storage of Canine Kidneys Using Sacks Solution, Transpl. 19:441-442, 1975). The basic difference that may account for this is the lack of adenosine and the low content of magnesium in the S-2 solution; as well as, the substitution of mannitol for glucose as the osmotic active substance. Mannitol may have a detrimental influence on the cells by causing structural changes, it is probably only temporary (Dahlager, J. I., and Bilde, T.: The Integrity of Tubular Cell Function After Preservation in Collins' or Sacks' Solution, Transpl. 21: 365-369, 1976). Moreover, the greater hyperosmolarity of the Sacks' solution (410-430 mOsm/kg) may greatly increase the tendency of mannitol and magnesium phosphate to precipitate out from solution within the kidney during cold storage (Collins, supra).
Moreover, since 1976, there have been numerous animal studies supporting the use of protective additives such as ATP-Mg Cl.sub.2 (Siegel, N. J., et al.: Enhanced Recovery of Renal ATP with Post Ischemic Infusion of ATP-Mg Cl.sub.2 Determined by 31P-NMR, Am. J. Physiol. 245:F530-534, 1983); (Stromski, M. E., et al.: Postischemic ATP-Mg Cl.sub.2 Provides Percursors for Resynthesis of Cellular ATP in Rats, Am. J. Physiol. 250:F834-F837, 1986); and, (Sumpio, B. E., et al.: Accelerated Functional Recovery of Isolated Rat Kidney with ATP-Mg Cl.sub.2 After Warm Ischemia, Am. J. Physiol. 247:R1047-R1053, 1984), AMP-Mg Cl.sub.2 (Stromski, M. E., et al.: Postischemic ATP-Mg Cl.sub.2 Provides Percursors for Resynthesis of Cellular ATP in Rats, Am. J. Physiol. 250:F834-F837, 1986) or inosine in flush or perfusate solutions. These additives are associated with improvement in post re-perfusion microcirculation and subsequent regeneration of intracellular ATP (Stromski, M. E., et al.: Chemical and Functional Correlates of Post Ischemic Renal ATP Levels, Proc. Natl. Acad. Sci. 83:6142-6145, 1986). It is postulated that these additives do not actually "recharge" the intracellular energy stores of renal cells, but they either slow the degradation of energy stores or supply substrate during the immediate re-perfusion period enabling prevention of cell swelling of intrarenal vascular endothelia cells, thus minimizing other aspects of the "reperfusion injury". Since hypothermia slows the ischemic induced degradation of ATP (Stromski, M. E., et al.: Chemical and Functional Correlates of Post Ischemic Renal ATP Levels, Proc. Natl. Acad. Sci. 83:6142-6145, 1986) and adenosine to hypoxanthine by a factor of 20, by adding ATP-Mg Cl.sub.2 or inosine this process may be slowed even more, generating even less hypoxanthine. With less hypoxanthine, less O.sub.2.sup.- is generated during re-perfusion and the cytochrome oxidase complex can now deal more efficiently with degradation of free radicals.
Furthermore, Belzer and his associates have recently developed a number of perfusates which demonstrate enhanced preservation properties. The improved post transplant microcirculation that was first noted in clinical renal transplant settings using Belzer perfusate (Belzer, F. O., et al.: A New Perfusate for Kidney Preservation, Transpl. Proc. 16:161-163, 1984) (containing ATP-Mg Cl.sub.2) in 1984 has been reproduced by many centers and has been associated with a significantly higher immediate function rate compared to perfusates containing silicate gel (Henry, M. L., Sommer, B. G., Ferguson, R. M.: Improved Immediate Function of Renal Allographs with Belzer Perfusate, Proc. American Society of Transplant Physicians, p. 70, 1987). However, the Belzer ATP-Mg Cl.sub.2 perfusate has not been used for simple storage, limiting its widespread use and value (Arch. Surg. 122: 790-794, 1987).
In addition, good results have also been recently reported through the use of a UW-1 solution produced by the University of Wisconsin (Wahlberg, J. A., Love, R., Landegaard, L., et al.: 72 hour Preservation of the Canine Pancreas, Transpl. 43:5-8, 1987); and (Ploeg, R. J., Goossens, D., McAnulty, et al.: Successful 72 Hour Storage of Dog Kidneys with UW Solution [submitted for publication]). The overall composition of UW-1 solution in comparison to the Collins' C-2, Sacks' S-2, and Belzer's Perfusate is set forth in Table 1. However, concerns over the possibility of allergic reaction from the high concentration of hydroxyethyl starch (50 gl/l) and the lack of FDA approval have slowed widespread availability and usage of the UW-1 solution.
As a result of the above indicated difficulties, the present inventors have conducted a great deal of experimental research in an attempt to produce a simple hypothermic intracellular flush and storage solution which enhances organ, particularly kidney, preservation both by diminution of reperfusion injury and by decreasing the loss of intracellular high energy metabolites that are necessary for viability. The present invention is the result of such experimental research.